The following background information is provided to assist the reader to understand the invention disclosed below and the environment in which it will typically be used. The terms used herein are not intended to be limited to any particular narrow interpretation unless clearly stated otherwise, either expressly or impliedly, in this document.
The heregulins (also called neuregulins, neu differentiation factor (NDF), acetylcholine receptor inducing activity (ARIA), and glial growth factors (GGFs)) are a family of growth factors that activate members of the ErbB/EGF receptor family (Holmes, Sliwkowski et al. 1992; Peles, Bacus et al. 1992; Wen, Peles et al. 1992; Falls, Rosen et al. 1993; Marchionni, Goodearl et al. 1993). Isoforms of heregulins, all of which arise from splice variants of a single gene, NRG-1 (neuregulin-1), have been cloned and classified into the α and β subgroups based on structural differences in their epidermal growth factor (EGF) like domains (Holmes, Sliwkowski et al. 1992).
ErbB-mediated signal transduction exerted by heregulins has been implicated in the regulation of diverse biological events including Schwann cell differentiation, neural regulation of skeletal muscle differentiation, heart development, and proliferation and differentiation of normal and malignant breast epithelial cells (Alroy and Yarden 1997; Sundaresan, Penuel et al. 1999). Research has shown that breast carcinoma cells respond to heregulin by activating signal transduction pathways that result in cellular proliferation, differentiation, as well as morphogenesis. Carcinoma cells expressing heregulin are typically hormone-independent and associated with the ability for metastasis in experimental studies.
ErbB3 is a transmembrane glycoprotein encoded by the c-erbB3 gene (Kraus, Issing et al. 1989; Plowman, Whitney et al. 1990). The ErbB3 receptor belongs to the ErbB family which is composed of four growth factor receptor tyrosine kinases, known as ErbB1/EGFR/HER1, ErbB2/Neu/HER2, ErbB4/HER4, as well as ErbB3/HER3. ErbB3 and ErbB4 are receptors for heregulins, whereas ErbB2 is a coreceptor (Carraway and Burden 1995). These receptors are structurally related and include three functional domains: an extracellular ligand-binding domain, a transmembrane domain, and a cytoplasmic tyrosine kinase domain (Plowman, Culouscou et al. 1993). The extracellular domain can be further divided into four subdomains (I-IV), including two cysteine-rich regions (II and IV) and two flanking regions (I and III). ErbB3 is unusual among these receptor tyrosine kinases in that its catalytic domain is defective. Despite its lack of intrinsic catalytic activity, ErbB3 is an important mediator of both heregulin and epidermal growth factor (EGF) responsiveness by way of its ability to heterodimerize with other ErbB family members, but especially with ErbB2. Heregulin binding induces ErbB3 to associate with other members of the ErbB family to form heterodimeric receptor complexes. ErbB3 then activates the kinase of its partner receptor which initiates a variety of c signaling cascades. ErbB3 also can relay information in response to diverse ligands, such as interferon and TNF-alpha, presumably through receptor cross-talk.
The ErbB3 receptor is important in regulating normal and aberrant cellular growth and differentiation, metastasis, and related pathologies. Transgenic mice that have been engineered to overexpress heregulin in mammary glands have been reported to exhibit persistent terminal end buds and, over time, to develop mammary adenocarcinomas (Krane and Leder 1996). ErbB3 expression studies on tumor tissues and on cancer cell lines show frequent co-expression of both ErbB2 and ErbB3 receptors (Alimandi, Heidaran et al. 1995; Meyer and Birchmeier 1995; Robinson, He et al. 1996; Siegel, Ryan et al. 1999). In addition, mammary tumors formed in transgenic mice that harbor an activated form of ErbB2 and metastasize to the lungs are associated with elevated amounts of tyrosine-phosphorylated ErbB2/Neu and ErbB3 (Siegel, Ryan et al. 1999). Many transformed cell lines used for experimental studies are either estrogen-dependent (MCF-7 and T47D, the low ErbB2 expressers) or estrogen-independent (SKBR3, high ErbB2 expressers). However, these cell lines do not exhibit metastatic phenotypes. When MCF-7 cells are transfected to overexpress ErbB2, MCF-7 cells gain an estrogen-independent phenotype, however, they never metastasize. On the other hand, MCF-7 cells that overexpress heregulin gain a metastatic phenotype, suggesting that the heregulins play an active role in metastasis (Hijazi, Thompson et al. 2000; Tsai, Hornby et al. 2000).
Five alternate ErbB3 transcripts arise from read-through of an intron and the use of alternative polyadenylation signals (Lee and Maihle 1998; Katoh, Yazali et al. 1993). Using 3′-RACE four novel c-erbB-3 cDNA clones of 1.6, 1.7, 2.1, and 2.3 kb from a human ovarian carcinoma-derived cell line have been isolated (Lee and Maihle 1998). p85-sErbB3 is encoded by a 2.1 kb alternate c-erbB3 transcript (cDNA clone R31F) that is translated into a 543 aa protein composed of subdomains I through III and the first third of subdomain IV of the ErbB3 extracellular domain, and a unique 24 amino acid carboxy-terminal sequence. p45-sErbB3 is encoded by a 1.7 kb alternate c-erbB3 transcript (cDNA clone R2F) that is translated into a 312 aa protein composed of subdomains I, II, and a portion of subdomain III of the extracellular domain of ErbB-3 followed by two unique glycine residues. p50-sErbB3 is encoded by a 1.6 kb alternate c-erbB3 transcript (cDNA clone R1F) that is translated into a 381 aa protein composed of subdomains I, II, and a portion of subdomain III of the extracellular domain of ErbB-3 followed by 30 unique amino acids. p75-sErbB3 is encoded by a 2.3 kb alternate c-erbB3 transcript (cDNA clone R35F) that is translated into a 515 aa protein composed of subdomains I through III, and has a unique 41 amino acid carboxy-terminal sequence (FIG. 1)(Lee and Maihle 1998).
A recombinant dominant-negative ErbB3 mutant with a deleted cytoplasmic domain but which retains its transmembrane domain can inhibit full-length ErbB2 and ErbB3 receptor phosphorylation and signal transduction (Ram, Schelling et al. 2000). In avian tissues, expression of a naturally occurring sEGFR/ErbB 1 inhibits TGFα dependent cellular transformation (Flickinger, Maihle et al. 1992). An aberrant soluble EGFR/sErbB1 secreted by the A431 human carcinoma cell line has been reported to inhibit the kinase activity of full-length EGFR in a ligand-independent manner (Basu, Raghunath et al. 1989). Similarly, herstatin, a naturally occurring soluble ErbB2 isoform which inhibits ErbB2 receptor phosphorylation and signaling, appears to function by blocking ErbB2 dimerization (Doherty, Bond et al. 1999). In no case do these soluble ErbB isoforms function as antagonists of ErbB receptor signaling through competitive, high affinity ligand binding.
Soluble ErbB3 proteins, for example p85-sErbB3 and p45-sErbB3, are unique among other naturally occurring soluble ErbB isoforms in that they bind specifically to heregulin with high affinity. Consequently, sErbB3 inhibits heregulin binding to cell surface ErbB receptors and heregulin-induced activation of the ErbB receptors and their downstream effectors. Thus sErbB3, such as p85-sErbB3 and p45-sErbB3, can be used as therapeutic reagents for heregulin-regulated malignancies such as mammary, prostate, ovary, and lung tumors. In addition, through receptor cross-talk, as well as through interactions with heterologous cell surface receptors, these soluble receptor isoforms may also be important for nonheregulin-associated conditions.